重组人白细胞介素—15的纯化及生物活性鉴定

目的探讨人白细胞介素15的纯化工艺和进行活性鉴定。方法将rhIL-15工程菌大量扩增后通过包涵体提取、复性、凝胶过滤、阴离子交换层析、高效反相液相色谱等技术进行纯化。结果经纯化得到rhIL-15,其分子量为14.4kD,蛋白纯度为99％以上,比活为1×107U/mg,对NK细胞杀伤活性具有明显的促进作用。结论通过三步纯化方法可以获得高纯度、高回收率和高生物学活性的rhIL-15,且具有明显的促NK细胞杀伤活性。     

人胎盘谷胱甘肽硫转移酶的简易纯化方法及抗血清制备

本文试用DEAE Cellulose_(52)离子交换柱层析代替高效液相层析的特异阴离子柱,模仿高效液相的梯度洗脱条件,成功地从人胎盘组织纯化制备出谷胱甘肽硫转移酶(GST),纯化863.1倍,比活性达16.4U/mg蛋白质,经SDS-PAGE鉴定,呈现单一亚基区带,亚基分子量为24 000。再用此酶作抗原研制出免抗人GST IgG,检测其纯度和特异性均得到满意的结果。     

肽核酸的研究与应用

肽核酸(PNA)是近年设计合成的一种不带磷酸戊糖骨架的寡核苷酸类似物。不同碱基寡聚体PNA均能与DNA和RNA的序列特异性靶位点结合,形成稳定的特殊结构,可用于不同的目的:①基因组定点切割、基因克隆与图谱分析;②基因表达调控;③基因突变检测。如果PNA能在细胞内发挥作用,将成为最理想的基因靶向药物。     

重阳木花粉过敏原的分析、鉴定与纯化

 【摘要】 目的　对重阳木花粉变应原蛋白进行分析、鉴定与纯化。方法　提取这重阳木花粉的粗提液,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS - PAGE)分离粗提液蛋白质组分并测定其分子量,收集过敏病人血清,采用免疫印迹(Western - blotting)法鉴定其变应原成分,通过离子交换层析对重阳木花粉变应原进行初步纯化和免疫印迹鉴定。结果　重阳木花粉有18条主要蛋白带,12 000Mr和14 000Mr为重阳木花粉特异性变应原;通过离子交换层析方法纯化出重阳木花粉分子量为12 000Mr和14 000Mr的变应原主要分布在II峰中。结论　对重阳木花粉变应原进行了初步的分离、鉴定和纯化,为临床重阳木花粉过敏疾病的诊断和治疗奠定了基础。     

多角度激光散射测定重组人核苷二磷酸激酶A在溶液中的聚集状态

目的和方法 :对重组人核苷二磷酸激酶A(rhNDPK -A)进行纯化 ,并对重组产物的理化性质及在溶液中的聚集状态进行测定。NDPK -A工程菌发酵后的菌体高压匀浆 ,然后微滤、超滤处理 ,所得样品经DEAE阴离子交换、Cibacronblue亲和层析、分子筛层析 3步纯化后 ,以SDS -PAGE和RP -HPLC分析纯化产物的纯度 ,RP -HPLC测定酶活性。合格制品以基质辅助激光解析飞行时间质谱测定分子量 ;Edman降解法测定N末端序列 ;多角度激光散射法测定重组产物在溶液中的表观分子量。结果 :rhNDPK -A纯化产物的SDS -PAGE纯度为 97 3% ,RP -HPLC纯…     

梭子蟹变应原的分离、纯化与鉴定

目的：对梭子蟹（Portunus pelogicus（Linnaeus））变应原进行分离,鉴定其主要及次要变应原,采用蛋白纯化技术获取梭子蟹天然的主要变应原并进行鉴定,为标准化变应原疫苗的研制提供理论依据.方法：取常规方法制备梭子蟹浸出液,经SDS-PAGE分离,测定各组分的相对分子量;同时用26例对蟹过敏的病人血清进行Western blot,鉴定其主要及次要变应原;利用快速制备液相色谱（FPLC）纯化技术（凝胶过滤层析和离子交换层析）获取主要变应原并作鉴定.结果：SDS-PAGE显示梭子蟹有19条可辨蛋白带,分子量在13 000～90 000之间,其中主带有9条,分子量是20 900、24 200、27 100、29 200、33 700、38 900、48 700、74 700、89 100;Western blot结果表明,26例蟹过敏患者血清全部呈阳性反应,浸出液中共有5条致敏条带,其中分子量在74 400、48 700的是主要变应原,阳性反应率均为100%;纯化后获取了74400、48700的主要变应原;经过免疫鉴定证实其具有免疫活性.结论：梭子蟹74 400和48 700的变应原为主要变应原,层析技术可以对分子量为74400和48700的主要变应原成分进行纯化.     

脂多糖结合蛋白的分离、纯化及其多抗的制备

目的 分离纯化大鼠脂多糖结合蛋白（LBP），并制备兔抗大鼠LBP多抗。方法 大鼠血清先通过硫酸铵盐析，再依次以Bio-Rex70阳离子交换层析和MonoQ阴离子交换层析进行纯化，纯化产物采用SDS－PAGE鉴定。用提纯的LBP免疫制备兔抗大鼠LBP的抗血清，用饱和硫酸铵盐析纯化后，经Westernblot鉴定其纯度。结果 分离纯化到较高纯度的LBP。用纯化到LBP免疫制备的兔抗LBP多抗与LBP具有良好的结合活性。结论 分离纯化到高纯度的LBP并制备出兔抗大鼠LBP多抗。     

HER-2/neu配体结合区2蛋白的甘露糖化修饰

目的 :建立一种蛋白质甘露糖化修饰的方法。方法 :用离子交换层析和钴亲和层析法 ,纯化重组HER 2 /neu配体结合区2 (LBD2 )蛋白 ,并将纯化后的LBD2蛋白进行甘露糖化修饰。新合成的LBD2拟糖蛋白经质谱法检测后 ,用间苯二酚 硫酸法进一步鉴定。结果 :纯化后LBD2蛋白的纯度可达 90 %。新合成的LBD2拟糖蛋白在质谱图上呈现相应于甘露糖修饰后蛋白质分子量的预期峰形。经间苯二酚 硫酸法检测 ,结合于LBD2蛋白上的化学基团为糖分子。结论 :获得了LBD2拟糖蛋白 ,为开展甘露糖受体介导的抗原提呈及肿瘤疫苗的实验研究打下了基础     

肠出血型大肠埃希菌O157：H7 EspF蛋白多克隆抗体的制备和初步纯化

目的制备肠出血型大肠埃希菌（EHEC）O157：H7EspF蛋白多克隆抗体并初步纯化。方法生物信息学方法分析EHECO157：H7EspF蛋白的柔韧性、亲水性、表面可能性及抗原表位,选取1条由15个氨基酸残基组成的多肽为半抗原,在其C端偶联钥孔血蓝蛋白（KLH）,免疫新西兰大白兔制备抗血清。ELISA测定抗血清效价,免疫印迹法鉴定其特异性,辛酸-硫酸铵法初步纯化抗血清,SDS-PAGE检测抗体纯度。结果选择EspF蛋白抗原指数最高的肽段72-TPSRPAPPPPTSGQA-86（0.506）为半抗原,合成抗原多肽经高效液相色谱鉴定,纯度为95.78%,经质谱分析其分子量为1563.76Mr,与目的多肽分子量一致;加强免疫3次后,EHECO157：H7EspF蛋白的抗血清效价达1：2048000;该血清对EHECO157：H7野生株和espF突变株（△espF）的EspF蛋白均有特异性,并经辛酸-硫酸铵法获得一定纯度的抗体。结论成功地制备了效价高、特异性强的EHECO157：H7EspF蛋白多克隆抗体。     

HPV16E7抗原HLA-A2限制性CTL表位预测及其合成

目的：预测、合成、纯化及鉴定人乳头瘤病毒E7抗原的HLA－A2限制性细胞毒性T淋巴细胞表位，为表位抗原特异性鉴定和临床研究提供靶肽。方法：采用超模体、多项式和量化模体方案相结合的方法，对靶抗原HPV16E7的HLA－A2限制性细胞毒性T淋巴细胞（Cytotoxic T lymphocyte,CTL)表位进行预测，并运用固相合成法合成多肽，经RP－HPLC纯化及纯度分析，用质谱进行定性鉴定。结果：分别预测出了16个、10个和3个九肽表位，确定其中5个九肽为候选合成表位，各合成肽的纯度都在95％以上。经质谱分析，各肽的分子量测定值与理论值相符。结论：超模体、多项式和量化模体方案联合应用可提高预测效率，避免了在研究E7抗原表位时盲目合成多肽；所合成的多肽为高纯度靶肽，可用于后续实验研究。     

In vitro bioactivity and osteoblast response of porous NiTi synthesized by SHS using nanocrystalline Ni-Ti reaction agent

Porous NiTi with an average porosity of 55 vol % and a general pore size of 100-600 microm was synthesized by self-propagating high temperature synthesis (SHS) with the addition of mechanically alloyed nanocrystalline Ni-Ti as the reaction agent. The SHS of porous NiTi using elemental powders was also performed for comparison. To enhance the bioactivity of the metal surface, porous NiTi synthesized by nanocrystalline Ni-Ti was subjected to chemical treatment to form a layer of TiO(2) coating. The porous NiTi with TiO(2) coating was subsequently immersed in a simulated body fluid (SBF) to investigate its apatite forming ability. The effects of the addition of nanocrystalline Ni-Ti as reaction agent and the application of apatite coating on osteoblastic behavior were studied in primary cultures of human osteoblast cells. Results showed that the main phases in porous NiTi synthesized by elemental powders were NiTi, Ti(2)Ni, and unreacted free Ni. By using nanocrystalline Ni-Ti as reaction agent, the secondary intermetallic phase of Ti(2)Ni was significantly reduced and the free Ni was eliminated. TiO(2) coating with anatase phase was formed on the surface of porous NiTi after the chemical treatment. A layer consisting of nanocrystalline carbonate-containing apatite was formed on the surface of TiO(2) coating after soaking in SBF. The preliminary cell culture studies showed that the porous NiTi synthesized with the addition of nanocrystalline Ni-Ti attracted marked attachment and proliferation of the osteoblast cells. This gives the evidence of the potential biomedical applications of the porous NiTi.     

Replication of tobacco mosaic virus RNA in tobacco mesophyll protoplasts inoculated in vitro

Synthesis of tobacco mosaic virus (TMV)-specific RNAs was investigated using tobacco mesophyll protoplasts inoculated in vitro. Three species of TMV-specific RNA were separated by polyacrylamide gel electrophoresis, and were identified as single-stranded viral RNA, its replicative form (RF), and replicative intermediate (RI) by examining their chromatographic behavior on cellulose, solubility in 1 M NaCl, and susceptibility to denaturation by dimethylsulfoxide. The molecular weight of RF was shown to be about 3.8 × 10⁶ by coelectrophoresis with rice dwarf virus RNA. Time course of the synthesis of RF and RI as well as the results of pulse-chase experiments were consistent with the possible precursor role of these structures in the synthesis of TMV-RNA. No other forms of TMV-specific RNA were detected in infected protoplasts. Kinetics of the replication of TMV-RNA in synchronously infected protoplasts showed the presence of three successive phases in virus replication. During the initial phase, viral RNA replicated exponentially and was encapsidated 4–5 hr later, so that most of the viral RNA synthesized remained free or only partially coated. The rate of viral RNA replication became linear at the end of the initial phase and remained so throughout the subsequent phases. Active formation of virus particles continued during the intermediate phase to encapsidate the bulk of viral RNA synthesized by this time. In the final phase, the synthesis of viral RNA was closely followed by encapsidation.     

Constructions of vaccinia virus A-type inclusion body protein,tandemly repeated mutant 7.5 kDa protein,and hemagglutinin gene promoters support high levels of expression

Summary We devised vaccinia virus (VV)-based vector systems that support higher levels of expression of cloned genes in the early and late phases of the infection cycle than reported previously. Enhanced expression can be obtained by combining the promoter of the A-type inclusion body protein gene, the mutated early region of the 7.5-kDa gene promoter (7.5-kDa promoter), and the promoter of the hemagglutinin (HA) gene. One construct produced 60 µg/10⁶ cells of chloramphenicol acetyltransferase (CAT), equivalent to 10–18% of total cell protein. Another construct produced about seven times more CAT during the early phase than the amount synthesized under the control of the 7.5-kDa promoter alone. The envelope proteins of human immunodeficiency virus type I synthesized during the early phase of infection were more active as immunogens than these proteins synthesized during the late phase, regardless of the amounts produced.     

Biodegradable pH/temperature-sensitive oligo(β-amino ester urethane) hydrogels for controlled release of doxorubicin

An injectable biodegradable pH/temperature-sensitive oligo(β-amino ester urethane) (OAEU) was synthesized. The OAEU was synthesized by addition polymerization between the isocyanate groups of 1,6-diisocyanato hexamethylene and the hydroxyl groups of a synthesized monomer piperazine dihydroxyl amino ester (monomer PDE) in chloroform in the presence of dibutyltin dilaurate as a catalyst. The synthesized OAEU was characterized by (1)H NMR spectroscopy, Fourier transform infrared spectroscopy and gel permeation chromatography. The aqueous solutions of OAEU showed a sol-to-gel-to-sol phase transition as a function of temperature and pH. The gel window covered the physiological conditions (37°C, pH 7.4) and could be controlled by changing the OAEU concentration. After a subcutaneous injection of the OAEU solution into Sprague-Dawley rats, a gel formed rapidly in situ and remained in the body for more than 2 weeks. The in vitro cytotoxicity test and in vitro degradation showed that the OAEU hydrogel was non-cytotoxic and biodegradable. The in vitro release of doxorubicin from this OAEU hydrogel was sustained for more than 10 days. This injectable biodegradable pH/temperature-sensitive OAEU hydrogel is a potential candidate as a drug/protein carrier and in biomedical applications.     

A mesophasic polymer: Poly(oxydodecandioyloxy-1,4-phenylene-2-methylvinylene-1,4-phenylene)

Poly(oxydodecandioyloxy-1,4-phenylene-2-methylvinylene-1,4-phenylene) ( 1 ) was synthesized and partially characterized by differential scanning calorimetry and X-ray diffraction methods. The polymer shows a thermotropic liquid crystalline behavior. The X-ray diffraction spectrum of the anisotropic liquid is coherent with a nematic phase. Solid state polymorphism is also observed.     

Replication of herpesvirus DNA. II. Sedimentation characteristics of newly synthesized DNA.

The sedimentation behavior of viral DNA synthesized during short labeling periods at various times after infection was investigated. These experiments indicated that viral DNA synthesis may be divided into the following two phases: (1) During the early phase, newly synthesized DNA is associated with structures which sediment with S values up to approximately twice that of mature viral DNA; (2) during the later phase, newly synthesized DNA is associated with structures that sediment much more rapidly. Both at early and later times after infection, approximately 20% of the newly synthesized DNA sediments in sucrose gradients more slowly than does mature viral DNA. Furthermore, after isopycnic centrifugation in CsCl, most of the newly synthesized viral DNA sediments in sucrose gradients more slowly than does mature viral DNA. The smaller than unit-size, newly synthesized viral DNA molecules are breakage products resulting from the fragility of newly synthesized DNA. These molecules fragment, not only because of their fragility in the regions of the replicative forks but also because of the presence of fragile sites at other positions along the newly synthesized DNA molecule. Experiments dealing with the transfer of parental DNA to progeny virions show that most parental viral DNA strands that are transferred to progeny virions retain their integrity. Breakage and reunion of parental viral DNA strands with progeny DNA is a relatively rare event.     

Synthesis of biomimetic Ca-hydroxyapatite powders at 37 degrees C in synthetic body fluids

An important inorganic phase for synthetic bone applications, calcium hydroxyapatite (HA, Ca10(PO4)6(OH)2), was prepared as a nano-sized (approximately 50 nm), homogeneous and high-purity ceramic powder from calcium nitrate tetrahydrate and diammonium hydrogen phosphate salts dissolved in modified synthetic body fluid (SBF) solutions at 37 degrees C and pH of 7.4 using a novel chemical precipitation technique. The synthesized precursors were found to easily reach a phase purity >99% after 6 h of calcination in air atmosphere at 90 degrees C, following oven drying at 80 degrees C. There was observed, surprisingly, no decomposition of HA into the undesired beta-TCP phase even after heating at 1,600 degrees C in air for 6 h. This observation showed the superior high-temperature stability of such 'biomimetic' HA powders as compared to those reported in previous studies. The former powders were also found to contain trace amounts of Na and Mg ions, originating from the use of SBF solutions instead of pure water during their synthesis. Characterization and chemical analysis of the synthesized powders were performed by X-ray powder diffraction, energy-dispersive X-ray spectroscopy, Fourier-transform infra-red spectroscopy, scanning electron microscopy, and inductively coupled plasma atomic emission spectroscopy.     

A Low-Temperature Thermoplastic Anti-bacterial Medical Orthotic Material Made of Shape Memory Polyurethane Ionomer: Influence of Ionic Group

PCL-based shape memory polyurethane ionomers with quaternarized pyridine moieties incorporated through molecular extension were synthesized. These polyurethanes were specifically designed as low-temperature thermoplastic anti-bacterial orthotic materials. A commercialized orthotic material was employed for comparison. The influence of ionic groups on the properties of orthotic materials was studied. The anti-bacterial properties and cytotoxicity of the polyurethane ionomer were tested. It was found that, different from other researchers' conclusions, the Coulombic force among cationic groups and the increased cohesion among hard segments after ionization could lead to polyurethane with better phase separation, soft segment phase crystallability and hard segment phase stability. The results of this work indicated that, although the ionic group had the function of improving phase separation, the asymmetric extender had a negative effect on the hard segment phase stability. Several of the main physical properties of the synthesized polyurethane as orthortic materials were studied. The materials mechanical properties at using temperature (ambient temperature 22°C) were improved so it may be used in circumstances where high mechanical strength is required. The fixity ratio was increased; as a result the material may fix shape more precisely according to patient affected parts. At last, a prototype wrist orthotic device was fabricated by hands. The orthotic device percent reductions against Klebsiella pneumoniae and Staphylococcus aureus were 96.2% and 100%, respectively. The anti-bacterial activity rating of this device was acceptable and significant according to ASTM E 2149. Cytotoxicity tests indicated that the wrist orthotic material was not cytotoxic.     

Matrix metalloproteinase-sensitive thermogelling polymer for bioresponsive local drug delivery

Development of a successful bioresponsive drug delivery system requires exquisite engineering of the materials so that they are able to respond to signals stemming from the physiological environment. In this study we propose a new Pluronic(?) based thermogelling system containing matrix metalloproteinase-2 (MMP2) responsive peptide sequences. A novel thermosensitive multiblock co-polymer comprising an MMP2-labile octapeptide (Gly-Pro-Val-Gly-Leu-Ile-Gly-Lys) was synthesized from a Pluronic(?) triblock co-polymer. The polymer was designed to form a thermogel at body temperature and degrade in the presence of MMP overexpressed in a tumor. The synthesized polymer was a multiblock co-polymer with ～2.5 U of Pluronic(?). The multiblock co-polymer solutions exhibited reverse thermal gelation around body temperature. The gelation temperatures of the multiblock co-polymer solutions were lower than those of the corresponding Pluronic(?) monomer at a particular concentration. The cytotoxicity of the synthesized polymer was lower compared with the monomer. The solubility of the hydrophobic anticancer drug paclitaxel was enhanced in the polymer solutions by micelle formation. The synthesized polymer was preferentially degraded in the presence of MMP. Paclitaxel release was dependent on the enzyme concentration. These findings suggest that the synthesized polymer has potential as a controlled drug delivery system due to its unique phase transition and bioresponsive behavior.